CN105738522A - Method for detecting squalene contained in leaf tobacco based on high performance liquid chromatography - Google Patents

Abstract

The invention discloses a method for detecting squalene contained in tobacco leaves based on high performance liquid chromatography. For tobacco leaf samples, chloroform was used as the extraction solvent, combined with Soxhlet extraction under scientific and reasonable process conditions to ensure a high extraction efficiency, and then purified by silica gel for detection by high-performance liquid chromatography. The invention further optimizes the detection conditions of the high-performance liquid chromatography, and the chromatographic peaks are well separated, and has the advantages of accuracy, rapidity, high sensitivity, good reproducibility, high recovery rate, etc., and not only meets the qualitative requirements, but also meets the quantitative requirements.

Description

A method for the detection of squalene in tobacco leaves based on high performance liquid chromatography

technical field

The invention relates to the technical field of detection of components in tobacco leaves, and more particularly relates to a method for detecting squalene contained in tobacco leaves based on high performance liquid chromatography.

Background technique

Squalene was first discovered from the liver oil of sharks and was named Squalene in 1914, and its chemical name is 2,6,10,15,19,23--hexamethyl--2,6,10,14, 18,22--Tetracosahexaene, which belongs to open-chain triterpenoids. Squalene has various physiological functions such as improving the activity of superoxide dismutase (SOD) in the human body, enhancing the body's antioxidant and immune capabilities, improving sexual function, anti-aging, anti-fatigue, and anti-tumor. It is a non-toxic Biologically active substances that have the functions of preventing and curing diseases. Recent studies have found that squalene also has a detoxifying effect on the human body, removing fat-soluble toxins from tissues. In April 2014, the State Tobacco Monopoly Administration issued the “Notice on Accelerating the Promotion and Application of Cigarette Harm Reduction Technology Achievements” (Guoyanbanzong [2014] No. 218), proposing to increase the major special research results of cigarette harm reduction technology To promote the application and promotion of cigarettes, targeted technical measures should be taken to speed up the pace of cigarette tar reduction and harm reduction.

The applicant's research work on the ecological accumulation and harm reduction of squalene, a natural antioxidant in tobacco leaves, found that squalene can selectively reduce harmful components such as benzopyrene, crotonaldehyde, and NH3 in smoke, and reduce the hazard index of cigarettes. At the same time, squalene has the function of removing gas-phase and solid-phase free radicals in smoke. Because squalene has unique physiological functions, the development and utilization of squalene has become a research hotspot in the scientific and technological circles.

However, there are currently no qualitative and quantitative detection methods for squalene in tobacco leaves that can be referenced at home and abroad. Therefore, it is very necessary and prospective to establish a qualitative and quantitative detection method for squalene in flue-cured tobacco in the tobacco industry.

Contents of the invention

The technical problem to be solved by the present invention is to fill in the deficiency of existing detection technology of squalene in tobacco leaves, and provide a method for detecting squalene contained in tobacco leaves based on high performance liquid chromatography.

Technical scheme of the present invention is realized by following technical scheme:

The present invention uses methanol and acetonitrile as the mobile phase, adopts the column chromatographic separation technology filled with a high-efficiency stationary phase with a particle size of 5 μm, optimizes the detection conditions of high-performance liquid chromatography, and injects the sample pretreated by the method of the present invention into a high-performance liquid chromatograph Carry out chromatographic separation, with ultraviolet absorption detector, detect the absorption value of squalene, adopt external standard method, calculate its content according to absorption peak area;

Specifically, a method for detecting squalene based on high performance liquid chromatography in tobacco leaves is provided, comprising the following steps:

S1. The sample solution is obtained by sample pretreatment, and the sample solution to be tested is obtained after silica gel purification;

S2. preparing a standard solution and establishing a standard curve;

S3. Detection: draw the sample to be tested and inject it into a high-performance liquid chromatograph for chromatographic separation, and use an ultraviolet absorption detector to detect squalene;

S4. adopt external standard method, calculate its content according to absorption peak area;

Wherein, the sample pretreatment described in step S1 includes the following steps:

S11. Tobacco leaves are cut into shreds, using chloroform as an extraction solvent, and the extract is obtained through Soxhlet extraction;

S12. Extract the extract with methanol: water with an extractant whose volume ratio is 70:30, collect the extract, perform rotary evaporation to nearly dryness, then dissolve it with acetonitrile, pass through a 0.22 μm organic membrane, and collect the filtrate to obtain a sample liquid;

S13. The sample liquid obtained in step S12 is purified through a small column, and the column is packed by a wet method, and the material of the small column is 0.06g of silica gel and an appropriate amount of anhydrous sodium sulfate;

The detection condition described in step S3 is:

Agilent1100 high performance liquid chromatography HPLC with UV detector;

Chromatographic column: C18, 5μm, 4.6×250mm;

Column temperature: 30°C;

Mobile phase type: methanol: acetonitrile, the volume ratio is 75/25;

Mobile phase flow rate: 1mL/min;

Detection wavelength: 210nm;

Injection volume: 10 μL.

Preferably, the method for preparing the standard solution described in step S2 is to accurately weigh 0.1g squalene standard substance, accurate to 0.0001g, dissolve it with acetonitrile and then settle to 100mL to prepare a 1000mg/L squalene standard solution, fully Shake well.

Preferably, the method for establishing the squalene standard curve described in step S2 is: using a serial dilution method, respectively preparing squalene standard solutions with a concentration of 0.002, 0.01, 0.05, 0.2, and 0.5 mg/mL in a high-performance liquid phase Chromatographic sampling detection; take the injection concentration as the abscissa and the peak area as the ordinate to establish a standard curve, and obtain the linear regression equation between the squalene concentration and the corresponding peak area.

Preferably, in step S11, before adding chloroform, the shredded tobacco is vortexed first, and the vortex treatment is vortexed for 1 min and then rested for 10 min.

Preferably, the condition of the vortex treatment is 2000r/min.

Preferably, the dosage of chloroform in step S11 is determined according to the ratio of 5.0 g shredded tobacco: 200 mL chloroform.

Preferably, the conditions of the Soxhlet extraction described in step S11 are water bath at 40°C and reflux for 9 hours.

Preferably, the amount of the extractant used in step S12 is determined according to the volume ratio of the extraction agent to the extraction solution is 1:1.

Preferably, the number of extractions in step S12 is 3 times.

In the present invention, adding an appropriate amount of anhydrous sodium sulfate to the purifying agent can well prevent the solution from washing the silica gel, resulting in unevenness. Preferably, the anhydrous sodium sulfate in step S13 is 0.5 g.

The present invention has the following beneficial effects:

Existing studies have shown that the flue gas after burning cigarettes contains nearly 5,000 compounds. According to the chemical analysis of the chloroform extract of tobacco leaves, there are more than 300 compounds in it, and the content of squalene is very low. For rapid and accurate detection, it is necessary to establish a scientific and systematic detection method. Aiming at tobacco leaf samples, the present invention establishes a high performance liquid chromatography method suitable for the detection of squalene in tobacco leaves, using chloroform as the extraction solvent, combined with the Soxhlet extraction method, to ensure a higher extraction efficiency, and then through silica gel The purification treatment of the column, under the technical conditions of the scientific pretreatment of the sample to be tested, further optimized the detection conditions, targeted to solve the problem of impurity peak treatment in the sample, and effectively utilized the accurate, fast and highly sensitive performance of high performance liquid chromatography. Advantages: It provides a detection method with better chromatographic peak separation, accuracy, speed, high sensitivity, good reproducibility and high recovery rate for the detection of squalene in tobacco leaves, which not only meets the qualitative requirements, but also meets the quantitative requirements.

Description of drawings

Fig. 1 squalene standard curve diagram.

Fig. 2 Chromatogram of squalene standard sample.

Figure 3 Chromatogram of unspiked target sample extracted with chloroform.

Fig. 4 Chromatogram of the added target sample extracted with chloroform.

Fig. 5 squalene (50mg/L) standard solution chromatogram.

Fig. 6 Background sample chromatogram.

Figure 7. Background added sample chromatogram.

Fig. 8 squalene standard solution (5mg/L) chromatogram.

Figure 9. Chromatogram of a sample without cleanup.

Figure 10 Chromatogram of a silica gel cleanup sample.

Figure 11 is a chromatogram of a sample purified by PSA (N-propylethylenediamine solid-phase adsorbent).

detailed description

The method of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The following embodiments and drawings are used for illustrative purposes only, and should not be construed as limiting the present invention. Unless otherwise specified, the reagent raw materials used in the following examples are commercially available or commercially obtained reagent raw materials. Unless otherwise specified, the methods and equipment used in the following examples are methods and equipment routinely used in the art. For the convenience of description, the instruments and reagents used in the examples of the present invention are described as follows, but the present invention is not limited thereby.

instrument:

(1) High-performance liquid chromatography HPLC (Agilent1100), equipped with: degassing pump, quaternary pump, automatic sampler, column thermostat and ultraviolet detector (Agilent Corporation of the United States)

(2) Shaker oscillator: SHZ-82AB type (Jiangsu Jintan City Ronghua Instrument Manufacturing Co., Ltd.)

(3) Platform scale: T-10000 type, d=0.1g (Shuangjie Brothers (Group) Co., Ltd. of the United States)

(4) Electronic balance: BSA224S type, d=0.1mg (Sartorius Scientific Instruments (Beijing) Co., Ltd.)

(5) Rotary vacuum evaporator: RE52-99 type (Shanghai Yarong Biochemical Instrument Factory)

(6) Vacuum filter pump: SHZ-DⅢ type (Gongyi City Yuhua Instrument Co., Ltd.)

(7) Ultrasonic cleaner: KQ-50 type (Kunshan Ultrasonic Instrument Co., Ltd.)

(8) Vortex instrument: MS3BS25 (IKA company)

(9) Constant temperature water bath: 501 digital display type (Honghua Instrument Factory, Jintan City, Jiangsu Province)

(10) All kinds of laboratory glassware

Reagent:

(1) 99.8% squalene reference substance (National Drug Control Institute)

(2) Methanol (chromatographically pure and analytically pure)

(3) Acetonitrile (chromatographically pure)

(4) n-Hexane (analytical pure)

(5) Chloroform (chloroform, analytically pure)

(6) Petroleum ether (analytical pure)

(7) 0.22μm organic microporous membrane (UK original membrane)

(8) Acetone (analytical pure)

(9) Cleaning agent: PSA, C18, silica gel (welchrom company)

(10) Shredded tobacco (Guangxi China Tobacco Industry Co., Ltd., randomly selected)

Example 1

The present embodiment provides a kind of detection method of squalene in tobacco leaf, comprises the following steps:

S1. Sample pretreatment to obtain the sample solution to be tested;

S2. preparing a standard solution and establishing a standard curve;

S3. Detection: draw the sample to be tested and inject it into a high-performance liquid chromatograph for chromatographic separation, and use an ultraviolet absorption detector to detect squalene;

S4. adopt external standard method, calculate its content according to absorption peak area.

Wherein, the sample pretreatment method described in step S1 is:

Sample pretreatment method: take 0.05mL, 10000mg/L squalene standard sample in 10mL n-hexane to obtain external standard base solution, after cutting tobacco leaves, take 5.0g shredded tobacco (take 0.05mL, 10000mg/L squalene standard sample In 10mL of n-hexane, then put 5.0g of shredded tobacco into it, vortex in a 2000r/min vortex for 1min, let it stand still, evaporate naturally to dryness (about 24h), and then wrap it with filter paper with a diameter of 15cm) in Soxhlet In the extractor, put 200mL of n-hexane, chloroform, and acetone into a round-bottomed flask respectively, put the Soxhlet extractor into a water bath at 40°C, and reflux for 9 hours to collect the extract. Rinse the shredded tobacco, combine the extracts, take the combined extracts and pass them through a small column, then rotatively evaporate to nearly dryness, then dissolve them with 5 mL of chromatographic grade acetonitrile, and pass through a 0.22 μm organic mesembrane to obtain the sample solution to be tested; The column is packed by wet method, and the column material is 0.06g silica gel + appropriate amount of anhydrous sodium sulfate;

The detection condition of described detection is:

High performance liquid chromatography HPLC (Agilent1100) with UV detector;

Chromatographic column: C18, 5μm, 4.6×250mm;

Column temperature: 30°C;

Mobile phase type: methanol: acetonitrile (volume ratio is 75/25);

Mobile phase flow rate: 1mL/min;

Detection wavelength: 210nm;

Injection volume: 10 μL.

The method for establishing the squalene standard curve described in step S2 is: adopt the serial dilution method, prepare the squalene standard solution that concentration is 0.002, 0.01, 0.05, 0.2, 0.5mg/mL respectively and inject in high performance liquid chromatography Detect; adopt external standard method to quantify, take injection concentration (mg/L) as abscissa, and peak area (UV S) is ordinate (shown in Table 1) and establish standard curve, as shown in accompanying drawing 1, find The linear regression equation of the squalene concentration and the corresponding peak area was obtained, and the regression equation was y=40.092x+52.046, R ² =0.9999.

Table 1 Corresponding relationship between squalene standard solution concentration and peak area

The results showed that the correlation coefficient of the standard curve was 0.9999, the linearity of the method was good, and it could be used for the determination of the detection limit and the calculation of the quantitative target.

Chromatogram of squalene standard sample, chromatogram of unadded target sample extracted with chloroform, chromatogram of added target sample extracted with chloroform, chromatogram of squalene (50mg/L) standard solution, background sample chromatogram, The chromatograms of the background added samples are shown in accompanying drawings 2 to 7 respectively.

Embodiment 2 Detection method of the present invention and conditional superiority verification test

For further verifying the superiority of detection method of the present invention and detection condition, carry out following experiment respectively:

2.1 Comparison of extraction efficiency of different extractants (C18 column)

2.1.1 Sample pretreatment method

Control group (CK): Take 5.0 g of shredded tobacco (wrapped with filter paper with a diameter of 15 cm) in a Soxhlet extractor, put 200 mL of each extraction solvent into a round-bottomed flask, and put the Soxhlet extractor into a 40°C water bath Reflux for 9 hours, collect the extract, take 30mL from the extract, put it into a separatory funnel, add 30mL of extract methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract The solution was evaporated to nearly dryness by rotary evaporation, then dissolved in 5 mL of chromatographic grade acetonitrile, passed through a 0.22 μm organic film, and then tested.

Add sample pre-treatment: Take 5.0g of shredded tobacco (take 0.05mL, 10000mg/L squalene standard sample in 10mL of n-hexane, then put 5.0g of shredded tobacco into it, vortex for 1min in a vortex instrument at 2000r/min, stand still, Evaporate naturally to dryness (about 24h), then wrap it with a filter paper with a diameter of 15cm) in a Soxhlet extractor, put 200mL of extraction solvents in a round bottom flask respectively, and put the Soxhlet extractor into a water bath at 40°C , reflux for 9 hours, collect the extract, take 30mL from the extract, put it into a separatory funnel, add 30mL of extract methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract , rotatively evaporated to nearly dryness, and then dissolved in 5 mL of chromatographic grade acetonitrile, passed through a 0.22 μm organic mesembrane, and stored at 4°C until testing.

To avoid repeating them one by one, this embodiment takes the extraction solvent n-hexane, chloroform or acetone as an example for illustration.

2.1.2 Results of comparison of extraction efficiency with different solvents

The results of the comparison of extraction efficiencies of different solvents are shown in Table 2. It can be seen from Table 2 that the extraction efficiency of chloroform is the highest.

The extraction efficiency comparison of different extractants in table 2

"-" in the table means "no addition or no recovery"

2.1.3 Discussion on the comparison results of different solvent extraction efficiencies

Using the Soxhlet extraction method, the efficiency of squalene in different extractants was compared. From the recovery rate of the results, it can be seen that the extraction efficiency of chloroform is the highest, reaching 62.27%, followed by n-hexane, reaching 36.36%. Acetone was the worst at 18.59%.

2.2 Comparison of different mobile phase ratios (C18 column)

In order to improve the analysis efficiency, in the experiment of this example, the influence of different ratios of mobile phase methanol: acetonitrile on the peak time was designed, including but not limited to: methanol: acetonitrile = 50:50, methanol: acetonitrile = 70:30, Methanol: Acetonitrile = 90:10, Methanol: Acetonitrile = 10:90, Methanol: Acetonitrile = 85:15, Methanol: Acetonitrile = 80:20, Methanol: Acetonitrile = 30:70, Methanol: Acetonitrile = 40:60, Methanol: Acetonitrile=75:25 and other different ratios. The peak eluting time of different ratios and the separation of the target peak and the impurity peak are shown in Table 3:

Table 3 Effects of different mobile phase ratios on the separation of target and impurity peaks

The best mobile phase conditions under optimized conditions (C18 column): methanol: acetonitrile = 75: 25 (30min) → methanol 100% (19min). Flow rate = 1 mL/min, detection wavelength = 210 nm, column temperature = 30°C.

Test result sees squalene (50mg/L) standard solution chromatogram, background sample chromatogram, background added sample chromatogram shown in accompanying drawing 5 to accompanying drawing 7.

When the ratio of the mobile phase is different, the separation effect of the target substance and the impurity peak in the sample is ideal when methanol: acetonitrile = 75:25.

2.3 Comparison of extraction efficiency of different extraction methods (C18 column)

Under the above-mentioned different optimized conditions, chloroform was used as the extractant for Soxhlet extraction, and the extraction efficiency of the sample was greatly improved when the extractant methanol-water (V:V=70:30) was used for extraction. The results are shown in Table 4:

Table 4 Comparison of extraction efficiency of target substance by different extraction methods

2.4 Comparison of purification effect of purifiers (C18 column)

2.4.1 Sample pretreatment method

Background sample pretreatment method (CK—silica gel): refer to the above treatment method, take 2mL of the extract after combining the extract, and carry out rotary evaporation, dissolve it with an appropriate amount of n-hexane, and then pass it through the column (0.06g of silica gel + 0.5g of Sodium sulfate water——wet washing column packing method), rotary evaporation to near dryness, dissolved in 2mL chromatography grade acetonitrile, passed through a 0.22μm organic filter membrane, and was to be tested. In the present invention, adding an appropriate amount of anhydrous sodium sulfate to the purifying agent can well prevent the solution from washing the silica gel, causing unevenness (the same below).

Sample pretreatment method (silica gel): Refer to the above treatment method, combine the extracts, take 2mL of a small column (0.06g silica gel + appropriate amount of anhydrous sodium sulfate----wet washing column method), rotary evaporate to nearly dry, use Dissolve in 2 mL of chromatographic grade acetonitrile, pass through a 0.22 μm organic filter membrane, and wait for the test.

Background sample pretreatment method (CK—straight): Refer to the above treatment method, combine the extracts and take 2mL of extracts for rotary evaporation to near dryness, dissolve in 2mL of chromatographic grade acetonitrile, pass through a 0.22μm organic filter membrane, and wait for the test.

Sample pretreatment method (straight): refer to the above treatment method, combine the extracts and take 2mL of rotary evaporation to near dryness, dissolve with 2mL of chromatographic grade acetonitrile, pass through a 0.22μm organic filter membrane, and wait for the test.

Background sample pretreatment method (CK—PSA): Refer to the above treatment method, take 2mL of the extract after combining the extract and carry out rotary evaporation, dissolve it with an appropriate amount of n-hexane, and then pass it through the column (0.06gPSA+0.5g anhydrous Sodium sulfate——wet wash column packing method), rotary evaporated to near dryness, dissolved in 2mL chromatographic grade acetonitrile, passed through a 0.22μm organic filter membrane, and was to be tested.

Sample pre-treatment method (PSA): refer to the above-mentioned treatment method, after combining the extracts, take a 2mL column (0.06gPSA + appropriate amount of anhydrous sodium sulfate----wet washing column method), rotary evaporate to near dryness, and use 2mL chromatographic Dissolve in grade acetonitrile, pass through a 0.22 μm organic filter membrane, and wait for the test.

2.4.2 HPLC conditions (C18 column)

Methanol: acetonitrile = 75:25 (30min) → methanol 100% (19min). Flow rate = 1 mL/min, detection wavelength = 210 nm, column temperature = 30°C.

Test result is shown in accompanying drawing 8 to accompanying drawing 11, and wherein, accompanying drawing 8 is squalene standard solution (5mg/L) chromatogram, accompanying drawing 9 is the chromatogram of sample without purification agent purification, and accompanying drawing 10 is The chromatogram of the sample purified by silica gel, and accompanying drawing 11 is the chromatogram of the sample purified by PSA. As can be seen from accompanying drawing 8 to accompanying drawing 11, the impurity peak in the sample purified by purifying agent is reduced a lot compared with unpurified, and the purifying effect of silica gel is better than the purifying effect of PSA simultaneously, so verified in the method of the present invention to determine to use silica gel Ideal as a purifier.

Claims (10)

1. one kind is detected the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography, it is characterised in that comprise the following steps:

S1. sample pre-treatments obtains sample liquid, obtains sample liquid to be measured after processing through silica gel purification；

S2. preparing standard solution, Criterion curve；

S3. detection: pipette samples liquid to be measured injects high performance liquid chromatograph and carries out chromatographic isolation, detects Squalene with ultraviolet absorption detector；

S4. external standard method is adopted, according to its content of absworption peak areal calculation；

Wherein, sample pre-treatments described in step S1 comprises the following steps:

S11. tobacco leaf chopping, using chloroform as Extraction solvent, obtains extracting solution through surname extraction；

S12. by extracting solution through methanol: the extractant that the volume ratio of water is 70:30 extracts, collect extract, rotate and be evaporated near doing, then dissolve with acetonitrile, cross 0.22 μm of organic mesentery, collect filtrate and obtain sample liquid；

S13. being processed by too small for step S12 gained sample liquid column purification, adopt wet method dress post, little column material is 0.06g silica gel and appropriate anhydrous sodium sulfate；

Described in step S3, the condition of detection is:

Agilent1100 high performance liquid chromatograph HPLC joins UV-detector；

Chromatographic column: C18,5 μm, 4.6 × 250mm；

Column temperature: 30 DEG C；

Mobile phase type: methanol: acetonitrile, volume ratio is 75/25；

Flow rate of mobile phase: 1mL/min；

Detection wavelength is: 210nm；

Sample size: 10 μ L.

2. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterized in that, the method of preparing standard solution described in step S2 is accurately to weigh 0.1g Squalene standard substance, it is accurate to 0.0001g, being settled to 100mL after dissolving with acetonitrile and be configured to the Squalene standard solution of 1000mg/L, fully vibration shakes up.

3. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterized in that, described in step S2, the method for the foundation of Squalene standard curve is: adopt serial dilutions, be configured to respectively concentration be 0.002,0.01,0.05,0.2, the Squalene standard solution of 0.5mg/mL is in high performance liquid chromatography sample detection；With sample introduction concentration for abscissa, peak area is vertical coordinate Criterion curve, obtains the equation of linear regression of Squalene concentration and respective peaks area.

4. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that tobacco shred, before adding chloroform, is first carried out vortex process by step S11, and it is vortex 1min that described vortex processes, static 10min.

5. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 4, it is characterised in that the condition that described vortex processes is 2000r/min.

6. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the consumption of chloroform described in step S11 is 5.0g tobacco shred according to the ratio with tobacco shred: 200mL chloroform is determined.

7. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the condition of described surname extraction described in step S11 is 40 DEG C of water-baths, refluxes 9 hours.

8. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the consumption of extractant described in step S12 is determined than for 1:1 with extracting liquid volume according to it.

9. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the number of times extracted described in step S12 is 3 times.

10. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that anhydrous sodium sulfate described in step S13 is 0.5g.